1. FIELD OF THE INVENTION
The enzyme glucose oxidase, which catalyzes the oxidation of glucose in the presence of oxygen to gluconic acid and hydrogen peroxide, is composed of the organic prosthetic group flavin adenine dinucleotide (FAD) conjugated to the protein portion of the enzyme, referred to as the apoenzyme. The protein portion, or apoenzyme, of glucose oxidase is referred to as apoglucose oxidase and is not enzymatically active when separated from FAD. The present invention relates to methods for preparing apoglucose oxidase, that is, for dissociating and separating FAD from the apoenzyme of glucose oxidase.
2. DESCRIPTION OF THE PRIOR ART
Apoglucose oxidase is useful as a reagent in specific binding assays which employ nonradioisotopic labels. U.S. Pat. application Ser. No. 917,961, filed June 22, 1978 and assigned to the present assignee, describes such a binding assay employing an organic prosthetic group as the label. A preferred organic prosthetic group label is FAD which can be monitored during the course of the assay based on its ability to combine with a corresponding apoenzyme, preferably apoglucose oxidase, to form a catalytically active holoenzyme.
An illustrative FAD-labeled specific binding assay employing apoglucose oxidase as a monitoring reagent comprises an initial step of placing the ligand to be determined, usually an antigen or hapten, in competition with an FAD-labeled form of the ligand for binding to a limited quantity binding partner, e.g., an antibody, of the ligand. The level of ligand present determines the proportion of the FAD-labeled ligand resulting in the so-called "bound" species, consituting FAD-labeled ligand bound to the binding partner, and the "free" species, consituting FAD-labeled ligand not so bound. The amount of FAD activity resulting in either of the bound- or free-species is therefore a function of the amount of ligand in the test sample. In the usual circumstance, the bound-species form of the FAD-labeled ligand, in comparison to its free-species form, has significantly decreased FAD activity as measured by its ability to combine with apoglucose oxidase to form an enzymatically active conjugate. Thus, upon introduction of apoglucose oxidase to the system, the amount of resulting glucose oxidase activity is a function of the amount of ligand in the test sample and can be readily determined by known spectrophotometric techniques.
Apoglucose oxidase is not commercially available and has been the subject of only a few technical publications. The prior art method of choice for preparing apoglucose oxidase is described by Swoboda in Biochim. Biophys. Acta 175:365-379(1969). The object of Swoboda's study was the relationship between molecular conformation and the binding of FAD in glucose oxidase. Aware that FAD is relatively strongly bound to the apoenzyme and cannot be removed by dialysis at neutral pH, Swoboda developed a method for dissociating and separating FAD and the apoprotein based on treatment with acidified ammonium sulfate.
In the Swoboda method, glucose oxidase is added dropwise at -5.degree. C. to saturated aqueous ammonium sulfate solution acidified to pH 1.4 with sulfuric acid. About 80% of the FAD is dissociated from the apoenzyme before the protein precipitates. After removal of the supernatant by centrifugation, the precipitate is redissolved and neutralized in sodium acetate solution. The neutralized solution is recycled several times through the acidifed salt treatment, centrifugation, and neutralization steps. The apoenzyme is then precipitated with saturated ammonium sulfate solution and again redissolved in buffer. Residual solids, including denatured protein, are removed by centrifugation yielding an aqueous apoenzyme preparation.
While the Swoboda method produces apoglucose oxidase useful for certain research purposes, the preparations obtained have significant residual glucose oxidase activity due to incomplete dissociation and separation of FAD. Residual enzyme activities of typical Swoboda preparations, expressed as a percent of glucose oxidase activity of the apoenzyme preparation in the presence of excess FAD, fall in the range of 1-10%, with some investigators reporting apoenzyme preparations with residual activities as high as 20% or more [Zappelli et al, Eur. J. Biochem 89:491-499(1978)].
The apoenzyme preparations obtained following the method of Swoboda have sufficiently low residual activities to permit a demonstration of the operability of FAD-labeled specific binding assays employing the apoenzyme as a monitoring reagent, however, the residual activities are undesirable from a commercial standpoint. Additional difficulties with the Swoboda method which arise in developing a commercial assay are the variable residual activities found from preparation to preparation and the cumbersome nature of the cycling treatments required. Accordingly, it is the object of the present invention to provide an improved method for preparing apoglucose oxidase whereby residual enzyme activities several times less than those obtained by the prior art methods are consistently produced.